Your browser doesn't support javascript.
: 20 | 50 | 100
1 - 20 de 205
Sci Rep ; 11(1): 23070, 2021 Nov 29.
Article En | MEDLINE | ID: mdl-34845256

HLA-G is a promiscuous immune checkpoint molecule. The HLA-G gene presents substantial nucleotide variability in its regulatory regions. However, it encodes a limited number of proteins compared to classical HLA class I genes. We characterized the HLA-G genetic variability in 4640 individuals from 88 different population samples across the globe by using a state-of-the-art method to characterize polymorphisms and haplotypes from high-coverage next-generation sequencing data. We also provide insights regarding the HLA-G genetic diversity and a resource for future studies evaluating HLA-G polymorphisms in different populations and association studies. Despite the great haplotype variability, we demonstrated that: (1) most of the HLA-G polymorphisms are in introns and regulatory sequences, and these are the sites with evidence of balancing selection, (2) linkage disequilibrium is high throughout the gene, extending up to HLA-A, (3) there are few proteins frequently observed in worldwide populations, with lack of variation in residues associated with major HLA-G biological properties (dimer formation, interaction with leukocyte receptors). These observations corroborate the role of HLA-G as an immune checkpoint molecule rather than as an antigen-presenting molecule. Understanding HLA-G variability across populations is relevant for disease association and functional studies.

Viruses ; 13(10)2021 Oct 19.
Article En | MEDLINE | ID: mdl-34696533

The Zika virus (ZIKV) has shown a promising oncolytic effect against embryonal CNS tumors. However, studies on the effect of different administration routes and the ideal viral load in preclinical models are highly relevant aiming for treatment safety and efficiency. Here, we investigated the effect and effectiveness of different routes of administration, and the number of ZIKVBR injections on tumor tropism, destruction, and side effects. Furthermore, we designed an early-stage human brain organoid co-cultured with embryonal CNS tumors to analyze the ZIKVBR oncolytic effect. We showed that in the mice bearing subcutaneous tumors, the ZIKVBR systemically presented a tropism to the brain. When the tumor was located in the mice's brain, serial systemic injections presented efficient tumor destruction, with no neurological or other organ injury and increased mice survival. In the human cerebral organoid model co-cultured with embryonal CNS tumor cells, ZIKVBR impaired tumor progression. The gene expression of cytokines and chemokines in both models suggested an enhancement of immune cells recruitment and tumor inflammation after the treatment. These results open new perspectives for virotherapy using the ZIKVBR systemic administration route and multiple doses of low virus load for safe and effective treatment of embryonal CNS tumors, an orphan disease that urges new effective therapies.

Front Immunol ; 12: 742881, 2021.
Article En | MEDLINE | ID: mdl-34650566

Despite the high number of individuals infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who develop coronavirus disease 2019 (COVID-19) symptoms worldwide, many exposed individuals remain asymptomatic and/or uninfected and seronegative. This could be explained by a combination of environmental (exposure), immunological (previous infection), epigenetic, and genetic factors. Aiming to identify genetic factors involved in immune response in symptomatic COVID-19 as compared to asymptomatic exposed individuals, we analyzed 83 Brazilian couples where one individual was infected and symptomatic while the partner remained asymptomatic and serum-negative for at least 6 months despite sharing the same bedroom during the infection. We refer to these as "discordant couples". We performed whole-exome sequencing followed by a state-of-the-art method to call genotypes and haplotypes across the highly polymorphic major histocompatibility complex (MHC) region. The discordant partners had comparable ages and genetic ancestry, but women were overrepresented (65%) in the asymptomatic group. In the antigen-presentation pathway, we observed an association between HLA-DRB1 alleles encoding Lys at residue 71 (mostly DRB1*03:01 and DRB1*04:01) and DOB*01:02 with symptomatic infections and HLA-A alleles encoding 144Q/151R with asymptomatic seronegative women. Among the genes related to immune modulation, we detected variants in MICA and MICB associated with symptomatic infections. These variants are related to higher expression of soluble MICA and low expression of MICB. Thus, quantitative differences in these molecules that modulate natural killer (NK) activity could contribute to susceptibility to COVID-19 by downregulating NK cell cytotoxic activity in infected individuals but not in the asymptomatic partners.

Asymptomatic Infections , COVID-19 , Histocompatibility Antigens , Major Histocompatibility Complex , SARS-CoV-2 , Adult , Aged , Brazil , COVID-19/genetics , COVID-19/immunology , Female , Genetic Predisposition to Disease , Genotype , Histocompatibility Antigens/genetics , Histocompatibility Antigens/immunology , Humans , Major Histocompatibility Complex/genetics , Major Histocompatibility Complex/immunology , Male , Middle Aged , Whole Exome Sequencing
J Bras Pneumol ; 47(4): e20200452, 2021.
Article En, Pt | MEDLINE | ID: mdl-34378644

OBJECTIVE: The shortage of viable lungs is still a major obstacle for transplantation. Trauma victims who represent potential lung donors commonly present hypovolemic shock leading to pulmonary inflammation and deterioration and rejection after transplantation. Seeking to improve lung graft, new approaches to donor treatment have been tested. This study focuses on treatment with mesenchymal stem cells (MSCs) or soluble factors produced by MSCs (FS-MSC) using a rat model for lung donors after hemorrhagic shock. METHODS: Forty-eight rats were divided into four groups: Sham (n=12), animals without induction of hypovolemic shock; Shock (n=12), animals submitted to hypovolemic shock (mean arterial pressure 40 mmHg); MSC (n=12), animals submitted to hypovolemic shock and treated with MSCs, and FS (n=12), animals submitted to hypovolemic shock and treated with FS-MSC. The animals were subjected to a 50-minute hypovolemic shock (40 mmHg) procedure. The treated animals were monitored for 115 minutes. We performed histopathology of lung tissue and quantification of inflammatory markers (TNF-α, IL-1ß, IL-6, IL-10, iCAM and vCAM) in lung tissue and peripheral blood leukocytes (PBLs). RESULTS: Hemorrhagic shock resulted in higher PBLs and neutrophil infiltrate in the lungs. FS animals had lower neutrophil density comparing with Shock and MSC animals (p<0.001). No differences in the cytokine levels in lung tissue were observed between the groups. CONCLUSIONS: The lungs of rats submitted to hemorrhagic shock and treated with FS-MSC showed reduced inflammation indicated in a decrease in lung neutrophil infiltrate.

Lung Transplantation , Mesenchymal Stem Cells , Shock, Hemorrhagic , Animals , Disease Models, Animal , Inflammation , Lung , Rats , Shock, Hemorrhagic/therapy
Int J Gynecol Pathol ; 2021 Aug 04.
Article En | MEDLINE | ID: mdl-34347668

In pregnant women, Zika virus (ZIKV) is associated with a congenital syndrome, most frequently involving damage to embryo brain formation and the development of microcephaly. The mechanism(s) by which ZIKV enters the maternal-fetal interface and is transmitted to the fetus remains incompletely determined. We sought to evaluate histologic changes in the placenta of ZIKV-infected pregnant women and to determine if this varied by maternal age. Placental samples were obtained from 66 women, 33 of whom were positive for ZIKV. Histologic evaluations were performed on 4 areas of the placenta: fetal surface, maternal surface, umbilical cord, and membranes. Samples were analyzed by the tissue microarray technique and tested for CD4, CD8, CD20, CD68, FOXP3, and cyclooxygenase-2 expression. Data were evaluated using Fisher exact test. ZIKV infection was more frequent in women less than 18 yr of age (9/11, 81.8%) than in women above 18 yr old (24/55, 43.6%) (P=0.0440). ZIKV detection was associated with neutrophilic chorioamnionitis (P=0.0332) and with septal (P=0.0244) and villous (P=0.0534) calcification. Hofbauer cell hyperplasia (P=0.0260) and cyclooxygenase-2 expression (P=0.0346) were more prevalent in ZIKV-positive women aged 18 yr and below than in the older ZIKV-positive women. ZIKV infection during pregnancy occurs more frequently in adolescents and induces higher rates of damage at the maternal-fetal interface than in older women.

Neuromuscul Disord ; 31(10): 1021-1027, 2021 10.
Article En | MEDLINE | ID: mdl-34404573

Sarcoglycanopathies are the most severe forms of autosomal recessive limb-girdle muscular dystrophies (LGMDs), constituting about 10-25% of LGMDs. The clinical phenotype is variable, but onset is usually in the first decade of life. Patients present muscle hypertrophy, elevated CK, variable muscle weaknesses, and progressive loss of ambulation. Four subtypes are known: LGMDR3, LGMDR4, LGMDR5 and LGMDR6, caused, respectively, by mutations in the SGCA, SGCB,SGCG and SGCD genes. Their four coded proteins, α-SG, ß-SG, λ-SG and δ-SG are part of the dystrophin-glycoprotein complex (DGC) present in muscle sarcolemma, which acts as a linker between the cytoskeleton of the muscle fiber and the extracellular matrix, providing mechanical support to the sarcolemma during myofiber contraction. Many different mutations have already been identified in all the sarcoglycan genes, with a predominance of some mutations in different populations. The diagnosis is currently based on the molecular screening for these mutations. Therapeutic approaches include the strategy of gene replacement mediated by a vector derived from adeno-associated virus (AAV). Pre-clinical studies have shown detectable levels of SG proteins in the muscle, and some improvement in the phenotype, in animal models. Therapeutic trials in humans are ongoing.

Int J Neonatal Screen ; 7(3)2021 Aug 11.
Article En | MEDLINE | ID: mdl-34449526

Since the approval of modifying therapies for Spinal Muscular Atrophy (SMA), several protocols aiming to screen SMN1 homozygous deletion in a neonatal context have been published. However, no work has compared different methodologies along with detailed implementation costs for centers where the neonatal screening of SMA has not yet been implemented. Therefore, our work compared different qualitative real-time PCR approaches for SMA screening and the estimated costs of test implementation. Using Brazilian blood samples, the presence and absence (P/A) and melt curve protocols were analyzed. MLPA was used as a confirmatory test. The costs were calculated for the simplex and multiplex tests plus equipment. The test workflow was based on the present experience and literature report. The accuracy of the P/A protocol was 1 (95% CI 0.8677-1) using dried blood spots (DBS). The melt curve protocol also achieved 100% concordance. The consumable costs ranged from USD 1.68 to 4.42 and from USD 2.04 to 12.76 per reaction, for the simplex and multiplex tests, respectively. The equipment acquisition costs ranged from USD 44,817.07 to 467,253.10, with several factors influencing this value presented. Our work presents a framework for decision-making, with a project demonstration of the different assays that will be useful in dealing with the issues of cost and availability of reagents. Moreover, we present a literature review and discussion of important concerns regarding treatment policies. We take the first step towards a future SMA NBS pilot program where it is not yet a reality.

Diagnostics (Basel) ; 11(8)2021 Aug 03.
Article En | MEDLINE | ID: mdl-34441334

Rapid diagnostics is pivotal to curb SARS-CoV-2 transmission, and saliva has emerged as a practical alternative to naso/oropharyngeal (NOP) specimens. We aimed to develop a direct RT-LAMP (reverse transcription loop-mediated isothermal amplification) workflow for viral detection in saliva, and to provide more information regarding its potential in curbing COVID-19 transmission. Clinical and contrived specimens were used to optimize formulations and sample processing protocols. Salivary viral load was determined in symptomatic patients to evaluate the clinical performance of the test and to characterize saliva based on age, gender and time from onset of symptoms. Our workflow achieved an overall sensitivity of 77.2% (n = 90), with 93.2% sensitivity, 97% specificity, and 0.895 Kappa for specimens containing >102 copies/µL (n = 77). Further analyses in saliva showed that viral load peaks in the first days of symptoms and decreases afterwards, and that viral load is ~10 times lower in females compared to males, and declines following symptom onset. NOP RT-PCR data did not yield relevant associations. This work suggests that saliva reflects the transmission dynamics better than NOP specimens, and reveals gender differences that may reflect higher transmission by males. This saliva RT-LAMP workflow can be applied to track viral spread and, to maximize detection, testing should be performed immediately after symptoms are presented, especially in females.

Am J Med Genet C Semin Med Genet ; 187(3): 357-363, 2021 09.
Article En | MEDLINE | ID: mdl-34189818

Diagnosis of individuals affected by monogenic disorders was significantly improved by next-generation sequencing targeting clinically relevant genes. Whole exomes yield a large number of variants that require several filtering steps, prioritization, and pathogenicity classification. Among the criteria recommended by ACMG, those that rely on population databases critically affect analyses of individuals with underrepresented ancestries. Population-specific allelic frequencies need consideration when characterizing potential deleteriousness of variants. An orthogonal input for classification is annotation of variants previously classified as pathogenic as a criterion that provide supporting evidence widely sourced at ClinVar. We used a whole-genome dataset from a census-based cohort of 1,171 elderly individuals from São Paulo, Brazil, highly admixed, and unaffected by severe monogenic disorders, to investigate if pathogenic assertions in ClinVar are enriched with higher proportions of European ancestry, indicating bias. Potential loss of function (pLOF) variants were filtered from 4,250 genes associated with Mendelian disorders and annotated with ClinVar assertions. Over 1,800 single nucleotide pLOF variants were included, 381 had non-benign assertions. Among carriers (N = 463), average European ancestry was significantly higher than noncarriers (N = 708; p = .011). pLOFs in genomic contexts of non-European local ancestries were nearly three times less likely to have any ClinVar entry (OR = 0.353; p <.0001). Independent pathogenicity assertions are useful for variant classification in molecular diagnosis. However, European overrepresentation of assertions can promote distortions when classifying variants in non-European individuals, even in admixed samples with a relatively high proportion of European ancestry. The investigation and deposit of clinically relevant findings of diverse populations is fundamental improve this scenario.

Genetic Variation , Genomics , Aged , Brazil , Exome , High-Throughput Nucleotide Sequencing , Humans
PeerJ ; 9: e11349, 2021.
Article En | MEDLINE | ID: mdl-33996288

Background: Type 2 diabetes mellitus (T2DM) and obesity are complex pandemic diseases in the 21st century. Worldwide, the T allele rs7903146 in the TCF7L2 gene is recognized as a strong GWAS signal associated with T2DM. However, the association between the C allele and obesity is still poorly explored and needs to be replicated in other populations. Thus, the primary objectives of this study were to evaluate the TCF7L2 rs7903146 association with T2DM according to BMI status and to determine if this variant is related to obesity and BMI variation in a cohort of elderly Brazilians. Methods: A total of 1,023 participants from an elderly census-based cohort called SABE (Saúde, Bem Estar e Envelhecimento-Health, Well-Being and Aging) were stratified by BMI status and type 2 diabetes presence. The TCF7L2 genotypes were filtered from the Online Archive of Brazilian Mutations (ABraOM-Online Archive of Brazilian Mutations) database, a web-based public database with sequencing data of samples of the SABE's participants. Logistic regression models and interaction analyses were performed. The BMI variation (∆BMI) was calculated from anthropometric data collected in up to two time-points with a ten-year-assessment interval. Results: The association between the rs7903146 T allele and T2DM was inversely proportional to the BMI status, with an increased risk in the normal weight group (OR 3.36; 95% CI [1.46-7.74]; P = 0.004). We confirmed the T allele association with risk for T2DM after adjusting for possible confound ing variables (OR 2.35; 95% CI [1.28-4.32]; P = 0.006). Interaction analysis showed that the increased risk for T2DM conferred by the T allele is modified by BMI (P interaction = 0.008), age (P interaction = 0.005) and gender (P interaction = 0.026). A T allele protective effect against obesity was observed (OR 0.71; 95% CI [0.54-0.94]; P = 0.016). The C allele increased obesity risk (OR 1.40; 95% CI [1.06-1.84]; P = 0.017) and the CC genotype showed a borderline association with abdominal obesity risk (OR 1.28; 95% CI [1.06-1.67]; P = 0.045). The CC genotype increased the obesity risk factor after adjusting for possible confounding variables (OR 1.41; 95% CI [1.06-1.86]; P = 0.017). An increase of the TT genotype in the second tertile of ∆BMI values was observed in participants without type 2 diabetes (OR 5.13; 95% CI [1.40-18.93]; P = 0.009) in the recessive genetic model. Conclusion: We confirmed that the rs7903146 is both associated with T2DM and obesity. The TCF7L2 rs7903146 T allele increased T2DM risk in the normal weight group and interacted with sex, age and BMI, while the C allele increased obesity risk. The TT genotype was associated with a lesser extent of BMI variation over the SABE study's 10-year period.

Hum Genome Var ; 8: 15, 2021.
Article En | MEDLINE | ID: mdl-33824725

SARS-CoV-2 utilizes the angiotensin-converting enzyme 2 (ACE2) receptor and transmembrane serine protease (TMPRSS2) to infect human lung cells. Previous studies have suggested that different host ACE2 and TMPRSS2 genetic backgrounds might contribute to differences in the rate of SARS-CoV-2 infection or COVID-19 severity. Recent studies have also shown that variants in 15 genes related to type I interferon immunity to influenza virus might predispose patients toward life-threatening COVID-19 pneumonia. Other genes (SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6, XCR1, IL6, CTSL, ABO, and FURIN) and HLA alleles have also been implicated in the response to infection with SARS-CoV-2. Currently, Brazil has recorded the third-highest number of COVID-19 cases worldwide. We aimed to investigate the genetic variation present in COVID-19-related genes in the Brazilian population. We analyzed 27 candidate genes and HLA alleles in 954 admixed Brazilian exomes. We used the information available in two public databases ( and and additional exomes from individuals born in southeast Brazil, the region of the country with the highest number of COVID-19 patients. Variant allele frequencies were compared with the 1000 Genomes Project phase 3 (1KGP) and gnomAD databases. We detected 395 nonsynonymous variants; of these, 325 were also found in the 1KGP and/or gnomAD. Six of these variants were previously reported to influence the rate of infection or clinical prognosis of COVID-19. The remaining 70 variants were identified exclusively in the Brazilian sample, with a mean allele frequency of 0.0025. In silico analysis revealed that seven of these variants are predicted to affect protein function. Furthermore, we identified HLA alleles previously associated with the COVID-19 response at loci DQB1 and DRB1. Our results showed genetic variability common to other populations and rare and ultrarare variants exclusively found in the Brazilian population. These findings might lead to differences in the rate of infection or response to infection by SARS-CoV-2 and should be further investigated in patients with this disease.

Front Genet ; 12: 636542, 2021.
Article En | MEDLINE | ID: mdl-33841501

We recently reported a deviation of local ancestry on the chromosome (ch) 8p23.1, which led to positive selection signals in a Brazilian population sample. The deviation suggested that the genetic variability of candidate genes located on ch 8p23.1 may have been evolutionarily advantageous in the early stages of the admixture process. In the present work, we aim to extend the previous work by studying additional Brazilian admixed individuals and examining DNA sequencing data from the ch 8p23.1 candidate region. Thus, we inferred the local ancestry of 125 exomes from individuals born in five towns within the Southeast region of Brazil (São Paulo, Campinas, Barretos, and Ribeirão Preto located in the state of São Paulo and Belo Horizonte, the capital of the state of Minas Gerais), and compared to data from two public Brazilian reference genomic databases, BIPMed and ABraOM, and with information from the 1000 Genomes Project phase 3 and gnomAD databases. Our results revealed that ancestry is similar among individuals born in the five Brazilian towns assessed; however, an increased proportion of sub-Saharan African ancestry was observed in individuals from Belo Horizonte. In addition, individuals from the five towns considered, as well as those from the ABRAOM dataset, had the same overrepresentation of Native-American ancestry on the ch 8p23.1 locus that was previously reported for the BIPMed reference sample. Sequencing analysis of ch 8p23.1 revealed the presence of 442 non-synonymous variants, including frameshift, inframe deletion, start loss, stop gain, stop loss, and splicing site variants, which occurred in 24 genes. Among these genes, 13 were associated with obesity, type II diabetes, lipid levels, and waist circumference (PRAG1, MFHAS1, PPP1R3B, TNKS, MSRA, PRSS55, RP1L1, PINX1, MTMR9, FAM167A, BLK, GATA4, and CTSB). These results strengthen the hypothesis that a set of variants located on ch 8p23.1 that result from positive selection during early admixture events may influence obesity-related disease predisposition in admixed individuals of the Brazilian population. Furthermore, we present evidence that the exploration of local ancestry deviation in admixed individuals may provide information with the potential to be translated into health care improvement.

Inquiry ; 58: 469580211007264, 2021.
Article En | MEDLINE | ID: mdl-33834861

OBJECTIVE: To evaluate the association between socioeconomic factors, health status, and Functional Capacity (FC) in the oldest senior citizens in a metropolis and a poor rural region of Brazil. METHOD: Cross-sectional study of 417 seniors aged ≥80 years, data collected through Brazil's Health, Well-being and Aging survey. FC assessed by self-reporting of difficulties in Activities of Daily Living (ADLs) and Instrumental Activities of Daily Living (IADLs). Chi-square tests and multiple logistic regression analyses were performed using "R" statistical software. RESULTS: Socioeconomic and demographic inequalities in Brazil can influence FC in seniors aged 80 years and older. Comparatively, urban long-lived people had a higher prevalence of difficulties for ADLs and rural ones showed more difficulties for IADLs. Among urban oldest seniors, female gender and lower-income were correlated with difficulties for IADLs. Among rural oldest seniors, female gender, stroke, joint disease, and inadequate weight independently were correlated with difficulties for ADLs, while the number of chronic diseases was associated with difficulties for IADLs. CONCLUSION: Financial constraints may favor the development of functional limitations among older seniors in large urban centers. In poor rural areas, inadequate nutritional status and chronic diseases may increase their susceptibility to functional decline.

Activities of Daily Living , Health Status Disparities , Brazil/epidemiology , Cross-Sectional Studies , Female , Humans , Socioeconomic Factors
Genet Mol Biol ; 44(1 Suppl 1): e20200302, 2021.
Article En | MEDLINE | ID: mdl-33651876

COVID-19 comprises clinical outcomes of SARS-CoV-2 infection and is highly heterogeneous, ranging from asymptomatic individuals to deceased young adults without comorbidities. There is growing evidence that host genetics play an important role in COVID-19 severity, including inborn errors of immunity, age-related inflammation and immunosenescence. Here we present a brief review on the known order of events from infection to severe system-wide disturbance due to COVID-19 and summarize potential candidate genes and pathways. Finally, we propose a strategy of subject's ascertainment based on phenotypic extremes to take part in genomic studies and elucidate intrinsic risk factors involved in COVID-19 severe outcomes.

Int J Obes (Lond) ; 45(5): 1017-1029, 2021 05.
Article En | MEDLINE | ID: mdl-33633342

BACKGROUND/OBJECTIVES: Admixed populations are a resource to study the global genetic architecture of complex phenotypes, which is critical, considering that non-European populations are severely underrepresented in genomic studies. Here, we study the genetic architecture of BMI in children, young adults, and elderly individuals from the admixed population of Brazil. SUBJECTS/METHODS: Leveraging admixture in Brazilians, whose chromosomes are mosaics of fragments of Native American, European, and African origins, we used genome-wide data to perform admixture mapping/fine-mapping of body mass index (BMI) in three Brazilian population-based cohorts from Northeast (Salvador), Southeast (Bambuí), and South (Pelotas). RESULTS: We found significant associations with African-associated alleles in children from Salvador (PALD1 and ZMIZ1 genes), and in young adults from Pelotas (NOD2 and MTUS2 genes). More importantly, in Pelotas, rs114066381, mapped in a potential regulatory region, is significantly associated only in females (p = 2.76e-06). This variant is rare in Europeans but with frequencies of ~3% in West Africa and has a strong female-specific effect (95% CI: 2.32-5.65 kg/m2 per each A allele). We confirmed this sex-specific association and replicated its strong effect for an adjusted fat mass index in the same Pelotas cohort, and for BMI in another Brazilian cohort from São Paulo (Southeast Brazil). A meta-analysis confirmed the significant association. Remarkably, we observed that while the frequency of rs114066381-A allele ranges from 0.8 to 2.1% in the studied populations, it attains ~9% among women with morbid obesity from Pelotas, São Paulo, and Bambuí. The effect size of rs114066381 is at least five times higher than the FTO SNPs rs9939609 and rs1558902, already emblematic for their high effects. CONCLUSIONS: We identified six candidate SNPs associated with BMI. rs114066381 stands out for its high effect that was replicated and its high frequency in women with morbid obesity. We demonstrate how admixed populations are a source of new relevant phenotype-associated genetic variants.

Mater Sci Eng C Mater Biol Appl ; 121: 111862, 2021 Feb.
Article En | MEDLINE | ID: mdl-33579511

Liver transplantation from compatible donors has been the main therapy available for patients with irreversible hepatic injuries. Due to the increasing shortage of organs suitable for transplantation, tissue engineering technologies are important alternatives or surrogate approaches for the future of human organ transplantations. New bioengineering tools have been designed to produce decellularized organs (i.e. scaffolds) which could be recellularized with human cells. Specifically, there is an unmet need for developing reproducible protocols for inducing better cellular spreading in decellularized liver scaffolds. The aim of the present work was to investigate the possibility to improve liver scaffold recellularization by pre-coating decellularized tissue scaffolds with HepG2-conditioned medium (CM). Furthermore, we evaluated the capability of commercial human liver cells (HepG2) to adhere to several types of extracellular matrices (ECM) as well as CM components. Wistar rat livers were decellularized and analyzed by histology, scanning electron microscopy (SEM), immunohistochemistry and residual DNA-content analysis. Human induced pluripotent stem cells (hiPSCs)-derived mesenchymal cells (hiMSCs), and human commercial hepatic (HepG2) and endothelial (HAEC) cells were used for liver scaffold recellularization with or without CM pre-coating. Recellularization occurred for up to 5 weeks. Hepatic tissues and CM were analyzed by proteomic assays. We show that integrity and anatomical organization of the hepatic ECM were maintained after decellularization, and proteomic analysis suggested that pre-coating with CM enriched the decellularized liver ECM. Pre-coating with HepG2-CM highly improved liver recellularization and revealed the positive effects of liver ECM and CM components association.

Induced Pluripotent Stem Cells , Proteomics , Animals , Culture Media, Conditioned/pharmacology , Extracellular Matrix , Humans , Liver , Rats , Rats, Wistar , Tissue Engineering , Tissue Scaffolds
Genet Med ; 23(4): 661-668, 2021 04.
Article En | MEDLINE | ID: mdl-33420346

PURPOSE: To identify novel genes associated with intellectual disability (ID) in four unrelated families. METHODS: Here, through exome sequencing and international collaboration, we report eight individuals from four unrelated families of diverse geographic origin with biallelic loss-of-function variants in UBE4A. RESULTS: Eight evaluated individuals presented with syndromic intellectual disability and global developmental delay. Other clinical features included hypotonia, short stature, seizures, and behavior disorder. Characteristic features were appreciated in some individuals but not all; in some cases, features became more apparent with age. We demonstrated that UBE4A loss-of-function variants reduced RNA expression and protein levels in clinical samples. Mice generated to mimic patient-specific Ube4a loss-of-function variant exhibited muscular and neurological/behavioral abnormalities, some of which are suggestive of the clinical abnormalities seen in the affected individuals. CONCLUSION: These data indicate that biallelic loss-of-function variants in UBE4A cause a novel intellectual disability syndrome, suggesting that UBE4A enzyme activity is required for normal development and neurological function.

Dwarfism , Intellectual Disability , Ubiquitin-Protein Ligases/genetics , Animals , Child , Developmental Disabilities/genetics , Humans , Intellectual Disability/genetics , Mice , Muscle Hypotonia , Phenotype , Syndrome , Whole Exome Sequencing
J Mol Neurosci ; 71(11): 2275-2280, 2021 Nov.
Article En | MEDLINE | ID: mdl-33464536

Myotonia congenita is a genetic disease caused by mutations in the CLCN1 gene, which encodes for the major chloride skeletal channel ClC-1, involved in the normal repolarization of muscle action potentials and consequent relaxation of the muscle after contraction. Two allelic forms are recognized, depending on the phenotype and the inheritance pattern: the autosomal dominant Thomsen disease with milder symptoms and the autosomal recessive Becker disorder with a severe phenotype. Before the recent advances of molecular testing, the diagnosis and genetic counseling of families was a challenge due to the large number of mutations in the CLCN1 gene, found both in homozygous or in heterozygous state. Here, we studied a consanguineous family in which three members presented a variable phenotype of myotonia, associated to a combination of three different mutations in the CLCN1 gene. A pathogenic splicing site mutation which causes the skipping of exon 17 was present in homozygosis in one very severely affected son. This mutation was present in compound heterozygosis in the consanguineous parents, but interestingly it was associated to a different second variant in the other allele: c.1453 A > G in the mother and c.1842 G > C in the father. Both displayed variable, but less severe phenotypes than their homozygous son. These results highlight the importance of analyzing the combination of different variants in the same gene in particular in families with patients displaying different phenotypes. This approach may improve the diagnosis, prognosis, and genetic counseling of the involved families.

Breast Cancer ; 28(2): 346-354, 2021 Mar.
Article En | MEDLINE | ID: mdl-32986223

BACKGROUND: It is estimated that 5-10% of breast cancer cases are hereditary. The identification of pathogenic germline variants allows individualized preventive health care, improvement of clinical management and genetic counseling. Studies in ethnically admixed Latin American populations have identified regions with increased frequency of deleterious variants in breast cancer predisposing genes. In this context, the Brazilian population exhibits great genetic heterogeneity, and is not well represented in international databases, which makes it difficult to interpret the clinical relevance of germline variants. METHODS: We evaluated the frequency of pathogenic/likely pathogenic (P/LP) germline variants in up to 37 breast cancer predisposing genes, in a cohort of 105 breast and/or ovarian cancer Brazilian women referred to two research centers between 2014 and 2019. RESULTS: A total of 22 patients (21%) were found to carry P/LP variants, and 16 VUS were detected in 15 patients (14.3%). Additionally, a novel pathogenic ATM intragenic deletion was identified in an early-onset breast cancer. We also detected a BRCA1 pathogenic variant (c.5074+2T>C) in higher frequency (10×) than in other studies with similar cohorts. CONCLUSIONS: Our findings contribute to the characterization of the genetic background of breast cancer predisposition in the Brazilian population as a useful resource to discriminate between deleterious variants and VUS, thus enabling improvement in the preventive health care and clinical management of carriers.

Ataxia Telangiectasia Mutated Proteins/genetics , Breast Neoplasms/genetics , Gene Deletion , Genetic Heterogeneity , Germ Cells/pathology , Germ-Line Mutation , Ovarian Neoplasms/genetics , Adult , Aged , BRCA1 Protein/genetics , BRCA2 Protein/genetics , Brazil/epidemiology , Breast Neoplasms/epidemiology , Cohort Studies , Female , Genetic Predisposition to Disease , Genetic Testing , Humans , Middle Aged , Ovarian Neoplasms/epidemiology , Young Adult